MULTI-ARRAY. 1-Plate Kit 5-Plate Kit 20-Plate Kit

Transcription

1 MESO SCALE DISCOVERY MULTI-ARRAY Assay System Human Leptin Kit 1-Plate Kit 5-Plate Kit 20-Plate Kit K151BYC-1 K151BYC-2 K151BYC-3 Meso Scale Discovery Meso Scale Discovery Meso Scale Discovery Meso Scale Discovery Meso Scale Discovery Meso Scale Discovery Meso Scale Di v4-2012Jul Page 1

2 MSD Metabolic Assays Human Leptin Kit This package insert must be read in its entirety before using this product. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. MESO SCALE DISCOVERY A division of Meso Scale Diagnostics, LLC Gaither Road Gaithersburg, MD USA MESO SCALE DISCOVERY, MESO SCALE DIAGNOSTICS, MSD, MSD (DESIGN), DISCOVERY WORKBENCH, QUICKPLEX, MULTI- ARRAY, MULTI-SPOT, SULFO-TAG, SECTOR, SECTOR HTS, SECTOR PR and 4 SPOT (DESIGN) are trademarks and/or service marks of Meso Scale Diagnostics, LLC Meso Scale Diagnostics, LLC. All rights reserved v4-2012Jul Page 2

3 Table of Contents I. Introduction... 4 II. Principle of the Assay... 4 III. Reagents Supplied... 5 IV. Required Material and Equipment not supplied... 5 V. Safety... 5 VI. Reagent Preparation... 6 VII. Assay Protocol... 7 VIII. Analysis of Results... 8 IX. Typical Standard Curve... 8 X. Sensitivity... 9 XI. Endogenous levels... 9 XII. Spike Recovery XIII. Linearity XIV. Assay Components XV. References Summary Protocol Plate Diagrams Ordering Information MSD Customer Service Phone: Fax: CustomerService@mesoscale.com MSD Scientific Support t a b l e o f c o n t e n t s o r d e r i n g i n f o r m a t i o n Phone: Fax: attn: Scientific Support ScientificSupport@mesoscale.com v4-2012Jul Page 3

4 I Introduction i n t r o d u c t i o n Leptin is a 16 kd product of the ob gene that is produced and released by adipocytes. Acting via cytokine-like receptors in the CNS, leptin plays a key role in metabolism and regulation of adipose tissue. Leptin is released in amounts mirroring overall body fat stores and acts on neurons and hypothalamic receptors thereby influencing the brain s perception of nutritional energy status and appetite. The absence of functional leptin (or its receptor) leads to uncontrolled food intake and resulting obesity. Fasting reduces circulating insulin and leptin levels in plasma. Leptin may therefore be a critical regulator of obesity often accompanied by insulin resistance and hyperinsulinemia. II Principle of the Assay p r i n c i p l e o f t h e a s s a y MSD metabolic assays provide rapid and convenient methods for measuring the levels of protein targets within single small-volume samples. The assays are available in both singleplex and multiplex formats. In a singleplex assay, an antibody for a specific protein target is coated on one electrode (or spot ) per well. In a multiplex assay, an array of capture antibodies against different targets is patterned on distinct spots in the same well. Our Human Leptin Assay detects leptin in a sandwich immunoassay (Figure 1). MSD provides a plate that has been pre-coated with leptin antibody. The user adds the sample and a solution containing the labeled detection antibody anti-leptin labeled with an electrochemiluminescent compound, MSD SULFO-TAG label over the course of one or more incubation periods. Leptin in the sample binds to capture antibody immobilized on the working electrode surface; recruitment of the labeled detection antibody by bound analyte completes the sandwich. The user adds an MSD read buffer that provides the appropriate chemical environment for electrochemiluminescence and loads the plate into an MSD SECTOR instrument for analysis. Inside the SECTOR instrument, a voltage applied to the plate electrodes causes the labels bound to the electrode surface to emit light. The instrument measures intensity of emitted light to afford a quantitative measure of leptin present in the sample. Leptin SULFO-TAG labeled detection antibody Analyte Insulin Capture antibody Figure 1. Sandwich immunoassay on MSD platform. The numbering convention for the different spots is maintained in the software visualization tools, on the plate packaging, and in the data files. Any spot that is not coated with a specific capture antibody is blocked with BSA to reduce non-specific binding to that spot. A unique bar code label on each plate allows complete traceability back to MSD manufacturing records v4-2012Jul Page 4

5 III Reagents Supplied r e a g e n t s s u p p l i e d Quantity per Kit Product Description Storage K151BYC-1 K151BYC-2 K151BYC-3 MULTI-SPOT 96-well Human Leptin, Insulin Plate(s) N45164A-1 SULFO-TAG Anti-hLeptin Antibody 1 (100X) Human Leptin Calibrator 10 µg/ml Blocker A Kit R93AA-2 (250 ml) Blocker E (100X) Diluent 6 R53BB-4 (8 ml) R53BB-3 (40 ml) R53BB-2 (200 ml) Diluent 12 R50JA-4 (10 ml) R50JA-3 (50 ml) Read Buffer T (4X) R92TC-3 (50 ml) R92TC-2 (200 ml) 2-8 C 1 plate 5 plates 20 plates 2-8 C <-70 C RT <-10 C <-10 C <-10 C RT 1 vial (40 µl) 1 vial (20 µl) (250 ml) 1 vial (0.09 ml) (8 ml) (10 ml) (50 ml) 1 vial (200 µl) 5 vials (20 µl ea) (250 ml) 1 vial (0.45 ml) (40 ml) (50 ml) (50 ml) 4 vials (200 µl ea) 20 vials (20 µl ea) 4 bottles (250 ml ea) 4 vials (0.45 ml ea) (200 ml ) 2 bottles (50 ml ea) (200 ml) IV Required Materials and Equipment - not supplied r e q u i r e d m a t e r i a l s a n d e q u i p m e n t n o t s u p p l i e d Deionized water for diluting concentrated buffers 50 ml tubes for reagent preparation 15 ml tubes for reagent preparation Microcentrifuge tubes for preparing serial dilutions Phosphate buffered saline plus 0.05% Tween-20 (PBS-T) for plate washing Appropriate liquid handling equipment for desired throughput, capable of dispensing 10 to 150 μl into a 96-well microtiter plate Plate washing equipment: automated plate washer or multichannel pipette Adhesive plate seals Microtiter plate shaker V Safety s a f e t y Safe laboratory practices and personal protective equipment such as gloves, safety glasses, and lab coats should be used at all times during the handling of all kit components. All hazardous samples should be handled and disposed of properly, in accordance with local, state, and federal guidelines. 1 Some SULFO-TAG labeled detection antibodies may be light-sensitive, so they should be stored in the dark v4-2012Jul Page 5

6 VI Reagent Preparation r e a g e n t p r e p a r a t i o n Bring all reagents to room temperature and thaw the Calibrator stock on ice. Blocker E can tolerate up to 5 freeze-thaw cycles. Alternatively, an aliquot of the blocker can be stored at 2-8 C for up to 1 month. Important: Upon first thaw, separate Diluent 6 and Diluent 12 into aliquots appropriate to the size of your assay needs. These diluents can go through up to three freeze-thaw cycles without significantly affecting the performance of the assay. Prepare Blocker A Solution Follow instructions included with the Blocker A Kit. Prepare Metabolic Assay Working Solution In a 15 ml tube combine (per plate): 70 µl of Blocker E 6930 µl of Diluent 6 Prepare Calibrator and Control Solutions Calibrator for the Human Leptin Assay is supplied at 10 µg/ml. For the assay, an 8-point standard curve is recommended with 3-fold serial dilution steps and a zero Calibrator. The table below shows the concentrations of the 8-point standard curve: To prepare this 8-point standard curve: Standard Leptin conc. Dilution (pg/ml) Factor Stock Cal. Vial STD STD STD STD STD STD STD STD-08 0 n/a 1) Prepare the highest Calibrator by transferring 10 µl of the Calibrator stock vial to 990 µl of Metabolic Assay Working Solution. 2) Prepare the next Calibrator by transferring 100 µl of the diluted Calibrator to 200 µl of Metabolic Assay Working Solution. Repeat 3-fold serial dilutions 5 additional times to generate 7 Calibrators. 3) The recommended 8 th Standard is Working Solution (i.e. zero Calibrator). 4) Diluted Calibrators should be kept on ice prior to addition to the plate. Note: The standard curve can be modified as necessary to meet specific assay requirements. Preparation of Serum and Plasma Samples The assay format requires 25 µl of sample per well. An adequate volume of each sample should be prepared depending upon desired number of replicates v4-2012Jul Page 6

7 Prepare Detection Antibody Solution The Detection Antibody is provided at 100X stock solution. The final concentration of the working Detection Antibody Solution should be at 1X. For each plate used, dilute a 30 µl aliquot of the stock Detection Antibody into 2.97 ml of Diluent 12. Prepare Read Buffer The Read Buffer should be diluted 4-fold in deionized water to make a final concentration of 1X Read Buffer T. Add 5 ml of 4X Read Buffer T to 15 ml of deionized water for each plate. Prepare MSD Plate This plate has been pre-coated with antibodies for the analytes shown in Figure 1. The plate can be used as delivered; no additional preparation (e.g., pre-wetting) is required. The plate has also been exposed to a proprietary stabilizing treatment to ensure the integrity and stability of the immobilized antibodies. VII Assay Protocol a s s a y p r o t o c o l 1. Addition of Blocker A Solution: Dispense 150 µl of Blocker A Solution into each well. Seal the plate with an adhesive plate seal and incubate for 1 hour with vigorous shaking ( rpm) at room temperature. 2. Wash and Addition of Sample or Calibrator: Wash the plate 3 times with PBS-T. Dispense 25 µl of Metabolic Assay Working Solution into each well of the MSD plate. Immediately add 25 µl of sample or Calibrator into the appropriate wells of the MSD plate. Seal the plate with an adhesive plate seal and incubate for 2 hours with vigorous shaking ( rpm) at room temperature. 3. Wash and Addition of the Detection Antibody Solution: Wash the plate 3 times with PBS-T. Dispense 25 µl of the 1X Detection Antibody Solution into each well of the MSD plate. Seal the plate and incubate for 1 hour with vigorous shaking ( rpm) at room temperature. 4. Wash and Read: Wash the plate 3 times with PBS-T. Add 150 µl of 1X Read Buffer T to each well of the MSD plate. Analyze the plate on the SECTOR Imager. Plates may be read immediately after the addition of Read Buffer. Notes Shaking a 96-well MSD MULTI-SPOT plate typically accelerates capture at the working electrode. Bubbles in the fluid will interfere with reliable reading of MULTI-SPOT plate. Use reverse pipetting techniques to insure bubbles are not created when dispensing the Read Buffer v4-2012Jul Page 7

8 VIII Analysis of Results a n a l y s i s o f r e s u l t s The Calibrators should be run in duplicate to generate a standard curve. The standard curve is modeled using least squares fitting algorithms so that signals from samples with known levels of the analyte of interest can be used to calculate the concentration of analyte in the sample. The assays have a wide dynamic range (3 4 logs) which allows accurate quantification in many samples without the need for dilution. The MSD DISCOVERY WORKBENCH analysis software utilizes a 4-parameter logistic model (or sigmoidal dose-response) and includes a 1/Y 2 weighting function. The weighting functionality is important because it provides a better fit of data over a wide dynamic range, particularly at the low end of the standard curve. IX Typical Standard Curve t y p i c a l s t a n d a r d c u r v e The MSD Human Leptin Assay is designed for use with human serum and plasma samples. The following standard curve is an example of the dynamic range of the assay. The actual signals may vary. A standard curve should be run for each set of samples and on each plate for the best quantification of unknown samples. Signal Leptin Concentration (pg/ml) Conc. (pg/ml) Average Signal Leptin %CV v4-2012Jul Page 8

9 X Sensitivity s e n s i t i v i t y The lower limit of detection (LLOD) is the calculated concentration of the signal that is 2.5 standard deviations over the zero Calibrator. The value below represents the average LLOD over multiple kit lots. LLOD (pg/ml) Leptin 43 XI Endogenous Levels E n d o g e n o u s l e v e l s Endogenous levels of human leptin in five matched individual serum and plasma samples. Sample Serum EDTA Plasma Heparin Plasma (pg/ml) (pg/ml) (pg/ml) Endogenous levels of human leptin in four diseased serum samples; BMI (Body Mass Index). Sample Leptin (pg/ml) Diabetes BMI Type I Type II Type II Type II v4-2012Jul Page 9

10 XII Spike Recovery s p i k e r e c o v e r y Serum, EDTA plasma, and heparin plasma were spiked with the Calibrators at multiple values throughout the range of the assay. Measured analyte represents average spike recovery in 4-6 pooled human serum and plasma samples. % Recovery = measured /expected x 100 Spiked Serum Spiked Heparin Plasma Spiked EDTA Plasma Spike Conc. (pg/ml) % Recovery XIII Linearity l i n e a r i t y Linearity was measured by spiking Calibrator levels in pooled human plasma followed by subsequent dilution. Percent recovery is calculated as the measured concentration divided by the concentration of the previous dilution (expected). % Recovery = measured x dilution factor / expected x 100 Fold Dilution % Recovery Serum EDTA Plasma Heparin Plasma v4-2012Jul Page 10

11 XIV Assay Components a s s a y c o m p o n e n t s Analyte Calibrator Human leptin Source Purified, recombinant human leptin expressed in E. coli Analyte Source Isoforms Recognized Species cross-reactivity Analyte Source Isoforms Recognized Species cross-reactivity Capture Antibody Human leptin Mouse monoclonal recognizes recombinant leptin and leptin circulating in human blood Human Detection Antibody Human leptin Mouse monoclonal n/a Human XV References r e f e r e n c e s 1. Matares G, Moschos S, Mantzoros CS. Leptin in Immunology. The Journal of Immunology, : Coll AP, Farooqi SI, O Rahilly S. The Hormonal Control of Food Intake Cell 129(2):l , Ahren B, Mansson S, Ginderich RL, Havel P. Regulation of plasma leptin in mice: influence of age, high-fat diet, and fasting. American Journal of Physiology (42): R113-R v4-2012Jul Page 11

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13 Summary Protocol MSD 96-well MULTI-ARRAY Human Leptin Kit MSD provides this summary protocol for your convenience. Please read the entire detailed protocol prior to performing the Human Leptin Assay. Step 1 : Sample and Reagent Preparation Bring all reagents to room temperature and thaw the Calibrator stock on ice. Prepare Blocker A Solution. Prepare serum or plasma samples. Prepare Metabolic Assay Working Solution and keep on ice. Prepare an 8-point standard curve using supplied Calibrator: The Calibrator should be diluted in Metabolic Assay Working Solution. Dilute the stock Calibrator 1:100 as indicated in Reagent Preparation section, then perform a series of 3-fold dilution steps and a no Calibrator blank. Diluted Calibrators should be kept on ice until use. Note: The standard curve can be modified as necessary to meet specific assay requirements. Prepare Detection Antibody Solution by diluting the 100X Anti-hLeptin Antibody to 1X in 3.0 ml of Diluent 12 per plate. Prepare 20 ml of 1X Read Buffer T by diluting 4X Read Buffer T with deionized water. Step 2 : Add Blocker A Solution Dispense 150 µl/well Blocker A Solution. Incubate at room temperature with vigorous shaking ( rpm) for 1 hour. Step 3 : Wash and Add Sample or Calibrator Wash plate 3 times with PBS-T. Dispense 25 µl/well Metabolic Assay Working Solution. Immediately, dispense 25 µl/well Calibrator or Sample. Incubate at room temperature with vigorous shaking ( rpm) for 2 hours. Step 4 : Wash and Add Detection Antibody Solution Wash plate 3 times with PBS-T. Dispense 25 µl/well 1X Detection Antibody Solution. Incubate at room temperature with vigorous shaking ( rpm) for 1 hour. Step 5 : Wash and Read Plate Wash plate 3 times with PBS-T. Dispense 150 µl/well 1X Read Buffer T. Analyze plate on SECTOR instrument v4-2012Jul Page 13

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